Recombinant human il 2 200 02

PBMCs derived from EBV carriers were resuspended at a concentration of 1 × 10⁶ cells/mL in R10 medium. Depending on the determined or known HLA types and the number of available cells, HLA-matching peptides and controls were added to individual wells. To prepare the stimulators, a third of the aliquot was washed in FCS-free RPMI-1640 media and incubated with single peptides (10 μg/mL) for 2 h, washed once, resuspended, and mixed with the remaining two-thirds of the cells, which were allocated as responders. Stimulators and responders were mixed and incubated for 3 days at 37 °C with 5% CO₂. On Day 3, the medium was partially replaced with one containing 25 U/mL recombinant human IL-2 (200-02, PeproTech, Waltham, MA, USA), and 20 ng/mL each of IL-7 (R&D, Minneapolis, MN, USA) and IL-15 (200-15, PeproTech), with half-media changes every two days. On Day 5, cells were stimulated again with autologous PBMCs preloaded with peptide. IL-2 was added to achieve a final concentration of 50 U/mL. After 14 days, peptide-specific CD8⁺ T cells were harvested for cell count and viability assessment.